EP1135689B1 - Stabilisation of calibrators containing cytokeratin - Google Patents

Abstract

The invention concerns calibrators or calibration solutions which are based on a human serum matrix and which are used in a method for detecting cytokeratin, a process for producing them, a method for stabilizing cytokeratin and an immunological method for detecting cytokeratin in a sample.

Description

The present invention relates to calibrators or calibration solutions based on based on a human serum matrix and which are used in a method for the detection of Cytokeratin are used, a process for their preparation, a process for Stabilization of cytokeratin in human serum and an immunological procedure for the detection of cytokeratin in a sample.

Immunological detection methods are of great importance in diagnostics obtained. They are characterized by high specificity and sensitivity, so that they too good for the detection of low concentration analytes in biological liquids are suitable. Immunological detection methods are particularly in the field of Infectious diseases, fertility and thyroid diagnosis Metabolic diseases and the diagnosis of major tumor diseases Importance. The most important tumor markers so far include, for example, carcinoembryonales Antigen (CEA), alpha fetoprotein (AFP), prostate specific antigen (PSA) and cytokeratins (CK).

Human cytokeratins are building blocks of the intermediate filaments that essentially do that Build the cytoskeleton of epithelial cells. More than 19 cytokeratins are known to exist which the cytokeratins 1 to 8 as basic and the cytokeratins 9 to 19 as acid Cytokeratins are called. The cytokeratins can become tetramers in the cell store together. A tetramer consists of two basic and two acidic cytokeratin molecules. Formed by linear aggregation of tetramers Filaments. Intact cytokeratin molecules are an integral part of the intermediate filament epithelial cells insoluble in water. The complexity and composition The cytokeratins are different in the different epithelial tissues, that is Epithelial cells have typical cytokeratin compositions for the respective tissue.

In tumor diagnostics, the soluble fragments of cytokeratin 19 are particularly important (CK 19) relevant, which are also referred to as CYFRA 21-1. One compared to healthy people increased concentration of CYFRA 21-1 indicates the presence a tumor disease. Up to now, elevated values have been found for bronchial carcinoma, Ovarian carcinoma, cervical carcinoma, bladder carcinoma and in head and neck tumors found. The main indication of CYFRA 21-1 is in the follow-up non-small cell bronchial carcinoma.

A method for the detection of CK 19 according to the principle of the sandwich ELISA is used described for example in EP-A-0 460 190. For this, the one to be examined Incubated a sample of a body fluid with at least two receptors R 1 and R2, where R1 and R2 are monoclonal antibodies, each different epitope of the CK 19 recognize. One of the antibodies is labeled with biotin, the other carries another Mark. The sandwich complex of R1, CK 19 and R2 binds to one with streptavidin coated solid phase. After separation of the solid from the liquid phase the marking in one of the two phases, preferably measured in the solid phase. Based of the signal obtained, the tumor marker CK 19 can be detected in the sample become.

When carrying out such a test, it is important to at least take the measured value obtained qualitative as positive (tumor marker is present) or negative (tumor marker is not available). This applies in particular to the classification of measurement data, which are obtained by means of automated systems. Often there is one Quantification of tumor marker concentrations desired or required. The Test system must therefore be carried out with reagents that have a defined value before carrying out the measurements Contain concentration of analyte, be calibrated. These defined reagents are referred to below as calibrators. The concepts of calibration solution, calibration standard, Standard solution or control are used synonymously for the term calibrator.

An important requirement for a calibrator is high stability. For one thing, must the accuracy of the test, a 100% recovery of the analyte in the Calibrator and a good signal-to-noise ratio can be ensured. The other must reliable reproducibility of the determination result over a longer period Period can be guaranteed. Therefore, calibrators must be used over a period of insensitive to their environmental conditions for several weeks, that is Temperature, direct sunlight on the laboratory table, pH value, buffer conditions etc. In unfavorable conditions there is a risk of hydrolysis, Proteolysis or denaturation of the calibrator. The use of a no longer intact Calibrators would lead to incorrect measurements.

Many tests are performed on serum samples. To compare the measurements To ensure, the calibrator should therefore also be based on a serum matrix his. The more sensitive the measuring system used, the larger they become Differences in measurement due to the jump in the matrix between sample and calibrator. It has been shown that cytokeratin on serum matrix is not stable. When storing a The cytokeratin-containing calibrator in the liquid state destroys the cytokeratin and denatured, so that the use of such a calibrator over a longer period Period is not possible.

In the past, the serum matrix was therefore replaced by an artificial matrix, which mimics the serum in terms of its composition. The artificial matrix is generally based on a buffer containing various salts and proteins (for example bovine serum albumin) is added to the natural environment Human serum with regard to salt and protein concentration as well as pH value as good as possible imitate. As a result, it was possible to add the analyte cytokeratin in the calibrator stabilize. A disadvantage of this approach is that it is not optimal Comparability, especially with sensitive measuring systems, since the samples Have human serum as the base matrix. So there is a jump in the matrix. During rehearsals in the low measuring range close to the cut-off value, this can lead to Incorrect measurements come. In extreme cases, this could lead to the fact that for the Art matrix-based calibrator did not determine a positive cytokeratin signal value with a value determined for a human serum sample, even if the analyte Cytokeratin is present in a comparable concentration.

The task was therefore to create a calibrator for methods for the detection of cytokeratin To provide, which is based on serum- preferably human serum matrix and one Period of at least several weeks, even under unfavorable environmental conditions is stable.

The task is solved by a calibrator, the essential components of which are serum, preferred are human serum, cytokeratin and aprotinin. It has turned out surprisingly shown that by adding the protease inhibitor aprotinin the cytokeratin can be effectively stabilized. It is therefore possible to use a calibrator for cytokeratin detection methods to be made available on a natural serum matrix basis and is produced in particular on the basis of a human serum matrix. This will be a matrix jump avoided when measuring the actual serum samples.

Furthermore, it has been shown that the calibrator according to the invention has a high long-term and Has temperature stability. The recovery rate for the in the calibrator is dissolved analyte cytokeratin, preferably CK 19, in an immunoassay after the Sandwich principle after three weeks of exposure to the lyophilized calibrator at 30 to 40 ° C and then redissolving almost 100%. So the cytokeratin becomes is not destroyed and is used by the antibodies used in the test even after exposure to time and temperature still recognized immunologically. The one at the beginning of the stability test measured concentrations of cytokeratin of the different calibrators correspond essentially the concentrations after the stress test. The stability and Precision of the calibrator according to the invention is thus comparable to the stability of the corresponding calibrator based on art matrix. Even when the calibrator is stored at 2 to 8 ° C in liquid or reconstituted form, which is the usual storage temperature of reagents in the refrigerator could fail even after 10 weeks Recovery of cytokeratin of almost 100% can be determined. That is, the The calibrator according to the invention is still in the refrigerator even after long storage without using qualitative or quantitative losses.

Crucial for the significantly improved stability of the calibrator according to the invention is the protease inhibitor aprotinin. Other protease inhibitors have proven not to be proven effective. Aprotinin is a commercially available Polypeptide consisting of 58 amino acids. It has an inhibitory effect on the coagulation factors XIIa, XIa and VIIIa as well as plasmin and plasma activators, also trypsin, Chymotrypsin and kallikrein. Surprisingly, other known protease inhibitors have emerged proven to be unsuitable for stabilizing the calibrator. Experiments with other substances such as detergents and salts have not desired stabilization effect.

Aprotinin is preferably in a concentration of at least 10 mg / l in the calibrator used. The maximum possible concentration is where the test interferes or where turbidity sets in due to the solubility that no longer exists. Especially concentrations between 25 and 40 mg / l are preferred.

The calibrator can be provided either in liquid form or as a lyophilisate become. To prepare the lyophilisate, all liquid and solid are first Components mixed or dissolved and then lyophilized. The Lyophilisate is then ultimately used as a reconstituted substance, i.e. again in liquid resolved, used. For reconstitution, that is, for redissolving the lyophilizate distilled water is generally used because it does not contain any unwanted ions added to the calibrator and in particular not adulterated the salt concentrations become. The amount of water depends on the desired cytokeratin concentration or the desired filling volume.

The calibrator according to the invention can also be conventional, known to those skilled in the art Contain substances such as salts or additional preservatives. For example N-methylisothiazolone and oxypyrion are preferred in the usual concentrations of about 1 mg / l each.

The calibrator according to the invention can preferably be in lyophilized form at 4 ° C. stored for several months without loss of quality. Storage can be done via a period of up to 36 months at 4 ° C. In reconstituted form the calibrator should be stored at 4 ° C for a few months.

Cytokeratin-free human serum is preferably used as the serum. This can be done by Processing can be obtained via affinity chromatography, or one of cytokeratin-free serum, which can be screened by appropriate methods must be determined.

a) Mischen von Serum mit Aprotinin

b) Filtrieren der Lösung

c) Lösen des Cytokeratins in Wasser

d) Mischen der Lösung aus Schritt b) mit dem gelösten Cytokeratin aus Schritt c)

e) Lyophilisieren der Lösungen aus d)

f) vor Gebrauch Lyophilisat in Wasser auflösen.

The invention also relates to a method for producing the calibrator according to the invention. The calibrator is preferably manufactured in the following steps

a) Mixing serum with aprotinin

b) filtering the solution

c) dissolving the cytokeratin in water

d) mixing the solution from step b) with the dissolved cytokeratin from step c)

e) lyophilizing the solutions from d)

f) Dissolve the lyophilisate in water before use.

If necessary, the pH in step d) can be adjusted to pH 7 to 8, preferably 7.2 become.

Another object of the invention is also a method for the production of stabilized cytokeratin, which is characterized in that preferred purified cytokeratin of the protease inhibitor aprotinin is added. Prefers the stabilized cytokeratin thus produced is used in a calibrator.

The invention also relates to the use of aprotinin for Stabilization of cytokeratin, especially CK 19.

The invention also relates to a method for stabilizing cytokeratin in serum, preferably human serum, which is characterized in that the serum Aprotinin, preferably in a concentration of at least 10 mg / l is added.

Another object of the invention is an immunological method for determination of cytokeratin in a sample, especially for the determination of CK 19. Das The method is characterized in that the signal intended for the sample with the A signal is compared, which is obtained with the aid of the calibrator according to the invention, the calibrator is measured using the same method as the sample.

a) Umsetzen der Probe mit einem für Cytokeratin spezifischen ersten Bindepartner, der eine mit einer Festphase bindefahige Gruppe trägt, über die die Bindung an eine Festphase erfolgen kann,

b) Umsetzen dieser Lösung mit einem weiteren Bindepartner, der eine Markierung trägt

c) Bindung des gebildeten Immunkomplexes an eine Festphase, wobei die Festphase bereits in Schritt a) vorhanden sein kann

d) Trennung der festen von der flüssigen Phase

e) Detektion der Markierung in einer oder beiden Phasen.

f) Vergleich der Messwerte des Kalibrators mit dem Wert der Probe und Quantifizierung.

Such a method for determining the cytokeratin and preferably CK 19 and the calibrator is preferably carried out in the following steps:

a) reacting the sample with a first binding partner specific for cytokeratin, which carries a group capable of binding to a solid phase, via which the binding to a solid phase can take place,

b) Implementing this solution with another binding partner who carries a label

c) binding of the immune complex formed to a solid phase, whereby the solid phase can already be present in step a)

d) separation of the solid from the liquid phase

e) detection of the marking in one or both phases.

f) Comparison of the measured values of the calibrator with the value of the sample and quantification.

The method can also be used as a competitive test according to the person skilled in the art Methods are carried out.

The binding partners are preferably mono- or polyclonal antibodies or their fragments such as F (ab ') ₂ , Fab' or Fab fragments, which specifically recognize and bind cytokeratin and in particular CK 19 immunologically. The antibodies are produced by methods familiar to the person skilled in the art. Also included are those antibodies which have been produced by changing the antibodies, for example by genetic engineering measures. The term antibody includes all of the meanings given above for binding partners.

The first binding partner specific for cytokeratin can either be directly linked to the Solid phase bound, or the binding to the solid phase takes place indirectly via a specific binding system. The direct binding of this binding partner to the solid phase takes place according to methods known to the person skilled in the art. If the bond is indirect via a specific binding system, the first binding partner is a conjugate, that of an antibody against cytokeratin and a reaction partner of one specific binding system. Be under a specific attachment system here understood two partners who can react specifically with each other. The Attachment can be based on an immunological reaction or on another specific reaction. Preferred is a specific binding system a combination of biotin and avidin or biotin and streptavidin used. Further preferred combinations are biotin and antibiotin, hapten and anti-hapten, Fc fragment of an antibody and antibodies against this Fc fragment or carbohydrate and lectin. One of the reaction partners of the specifically bindable pair is then Part of the conjugate.

The other reaction partner of the specific binding system for the first binding partner is available as a coating of the solid phase. Binding the other reactant of the specific binding system to an insoluble carrier material be carried out according to the usual methods known to those skilled in the art. in this connection Both covalent and adsorptive binding are suitable.

Test tubes or microtiter plates made of polystyrene or are suitable as the solid phase Similar plastics, which are on the inner surface with a reaction partner of the specific binding system are coated. Also suitable and particularly preferred are particulate substances, such as latex particles, magnetic Particles, molecular sieve materials, vitreous bodies, plastic tubes and like. Porous, layered supports such as paper can also be used as supports become. Magnetic beads, so-called beads, are particularly preferred used, which in turn with the corresponding binding partner of the above specific binding system are coated. These microparticles can then Sequence of the test reaction for carrying out the detection reaction, for example, by Filtration, centrifugation or in the case of magnetic particles by a magnet be separated from the liquid phase.

Detection of specific binding reactions between antibodies against Cytokeratin and cytokeratin can be done in different ways. Generally is a binding partner marked the specific binding reaction. Usual markings are Chromogens, fluorophores, substances capable of chemi- or electrochemiluminescence, Radioisotopes, haptens, enzyme labels or substances, which in turn a can form a specific binding pair such as, for example, biotin / streptavidin.

As samples for performing the method for the detection of cytokeratin can all biological liquids familiar to the person skilled in the art are used. Prefers body fluids such as whole blood, blood serum, blood plasma, urine or Saliva, particularly preferably blood serum used.

The following examples further illustrate the invention.

example 1 Production of the calibrator according to the invention

A: Humanserum, käuflich erworben, affinitätschromatographisch cytokeratin-frei gemacht

B: Aprotinin 34 mg, Id.-Nr.: 0236632-001

C: N-Methylisothiazolon-HCL 1g, Id.-Nr.: 1085901-105

D: Oxypyrion 1g, Id.-Nr.: 1085913-05

E: Cytokeratin 7,31 g aus einer Stocklösung mit der Konzentration von 0.05 µg/ml mit 941 g A+B+C+D

The specified identification numbers (Id.-Nr.) designate catalog numbers from the catalog of Boehringer Mannheim GmbH, Germany. To make 1 liter CK calibrator (CK concentration 40 ng / ml), mix together:

A: Human serum, purchased, made cytokeratin-free using affinity chromatography

B: Aprotinin 34 mg, ID no .: 0236632-001

C: N-methylisothiazolone HCL 1g, ID no .: 1085901-105

D: Oxypyrion 1g, ID no .: 1085913-05

E: Cytokeratin 7.31 g from a stock solution with a concentration of 0.05 µg / ml with 941 g A + B + C + D

The human cytokeratin Cyfra 21-1 used corresponds to the cytokeratin from the human cell line MCF-7 according to Cyfra 21-1 CalSet, calibration set for the Elecsys® CYFRA 21-1 Immunoassay from Boehringer Mannheim GmbH Germany, ID no. 1,820,974th

Example 2 Calibrator load at 35 ° C

The process is carried out according to Boehringer's Elecsys® Cyfra 21-1 process Mannheim GmbH (Id.No. 1 820 966). The Elecsys® test procedure is based on biotin / streptavidin technology. The test principle is a 1-step sandwich ELISA with two Antibodies that recognize different epitopes on the analyte (cytokeratin). The Solid phase is formed by magnetic latex beads coated with streptavidin which binds a biotinylated antibody specific for cytokeratin. The detection of the bound tumor marker takes place after the separation of the solid from the liquid phase by measuring electrochemiluminescence from a second for cytokeratin specific antibodies labeled with a ruthenium complex.

R1 (erster Antikörper, biotinyliertes Fab-Fragment): MAK<CK19>M-KS19.1-Fab(DE)-Bi(DDS); Konzentration 1,5 µg/ml

R2 (zweiter Antikörper, ruthenyliertes IgG): MAK<CK19>M-BM19.21-IgG-BPRU); Konzentration 2,1 µg/ml

The following ingredients are used:

R1 (first antibody, biotinylated Fab fragment): MAK <CK19> M-KS19.1-Fab (DE) -Bi (DDS); Concentration 1.5 µg / ml

R2 (second antibody, ruthenylated IgG): MAK <CK19>M-BM19.21-IgG-BPRU); Concentration 2.1 µg / ml

90 µl R1 and 90 µl R2 are coated with streptavidin-coated magnetic beads according to the manufacturer's instructions for the Elecsys® 2010 device from Boehringer Mannheim GmbH, Germany, incubated and measured together.

The cytokeratin-containing calibrator according to the invention, which was freshly dissolved, and the calibrator according to the invention, which had been subjected to a lyophilized state at 35 ° C. for three weeks and then reconstituted, are compared. The results are shown in Table 1. Human matrix master calibrators, freshly dissolved and after 3 weeks of exposure at 35 ° C freshly solved after 3 weeks of exposure at 35 ° C MK Conc. (Ng / ml) Wdf. (%) Conc. (Ng / ml) Wdf. (%) too fresh 1 0.0 / 0.0 / 2 0.88 100 0.87 99 3 7.57 100 7.10 94 4 18.13 100 17.19 95 5 60.85 100 57.62 95 6 125 100 119 95 7 302 100 296 98 8th 450 100 430 96 9 613 100 604 98 MK: Master Calibrator: Calibrator used to calibrate a new batch of reagents before delivery
Wdf: recovery
bel: burdened

It turns out that the loaded calibrator also has a good, almost 100% recovery shows. The deviation is less than 10% (largest deviation: 6%). The The calibrator according to the invention is therefore above a high temperature stable for a longer period of time and provides reliable calibration values even after loading. Even if the calibrator is inadvertently incorrectly stored, it can be used without hesitation can be used without any loss of quality.

Example 3 Long-term exposure of the calibrator at 4 ° C (liquid)

A comparison is made between the freshly dissolved calibrator and the calibrator, which was stored as a reconstituted liquid at 4 ° C for 10 weeks before the measurement. The measurements are carried out as described in Example 2. The results are shown in Table 2. Human matrix standards, freshly dissolved and after 10 weeks of storage at 4 ° C freshly solved after 10 weeks of storage at 4 ° C default Conc. (Ng / ml) Wdf. (%) Conc. (Ng / ml) Wdf. (%) too fresh A 0.20 / 0.44 / B 3.33 100 3.13 94 C 8.00 100 7.85 98 D 18.84 100 17,81 95 e 54.52 100 51.68 95 F 100.89 100 95.53 95

The calibrator according to the invention also shows itself in liquid form after exposure good results regarding signal and concentration recovery over 10 weeks at 4 ° C.

Example 4 Long-term stability of the calibrator: 24 months at 4 ° C / 24 months - 20 ° C

The calibrator that was stored at 4 ° C for 24 months before the measurement is compared with the calibrator that was stored at -20 ° C for 24 months (each as a lyophilisate). The procedure is as described in Example 2. The results are shown in Table 3. Human matrix calibrators after storage for 24 months at -20 ° C and +4 ° C 24 months at -20 ° C 24 months at +4 ° C default Conc. (Ng / ml) Wdf. (%) Conc. (Ng / ml) Wdf. (%) too fresh MK 1 0.0 / 0.0 / MK 2 3.71 100 3.85 104 MK 3 9.01 100 9.04 100 MK 4 40.11 100 39.82 99 MK 5 394.8 100 395.0 100

There is good recovery for both stress methods. The cytokeratin-containing Calibrator can be used in lyophilized form without concern over periods of time Years at + 4 or - 20 ° C without the quality suffering significantly.

Example 5 Comparison of the measured values from tumor marker controls: Calibration of the measuring device based on art matrix and human serum

Before carrying out the measurements, the Elecsys® measuring device with the cytokeratin calibrated calibrator based on art matrix and then the tumor marker controls measured on the basis of these calibration values. The ones found for this measurement By definition, values are set to 100% recovery. Then will calibrated the same device with the calibrator based on human serum according to the invention and again measure the tumor marker controls using these calibration values. The Results are shown in Table 4.

The tumor marker controls TMK I or II and PCT I or II are based on processed human serum and contain, in addition to other tumor markers such as PSA and AFP, the cytokeratin Cyfra 21-1 with target values for two concentration ranges (I and II). Comparison between tumor marker controls; Calibration for cytokeratin based on synthetic matrix compared to calibration based on human serum Calibration with calibrator based on synthetic matrix Calibration with human serum based calibrator Tumor marker-control Conc. (Ng / ml) Wdf. (%) Conc. (Ng / ml) Wdf. (%) TMKI 4.63 100 5.07 110 TMK II 28.5 100 29.8 105 PCTI 4.99 100 5.23 105 PCT II 30.7 100 31.7 103

It can be shown that the deviations for the measurements of the tumor marker controls based on the calibrations with the calibrators based on art or human matrix amount to a maximum of 10%. The ones obtained for tumor marker controls Values are therefore independent of the one used for the calibration of the device Calibrator. The calibrator based on human serum can therefore be the calibrator based on art matrix replace.

Claims (9)

1. Calibrator for a method to detect cytokeratin in a sample containing serum, cytokeratin and aprotinin.
2. Calibrator as claimed in claim 1, wherein human serum is used as the serum.
3. Calibrator as claimed in claim 1, wherein the added cytokeratin is CK 19.
4. Calibrator as claimed in claim 1, wherein it contains aprotinin at a concentration of at least 10 mg per litre.
5. Process for producing a calibrator as claimed in claim 1 comprising the steps:

   a) mixing serum with aprotinin

   b) filtering the solution

   c) dissolving the cytokeratin in water

   d) mixing the solution from step b) with the dissolved cytokeratin from step c)

   e) lyophilizing the solutions from d)

   f) dissolving the lyophilisate in water before use.
6. Method for stabilizing cytokeratin in serum, wherein aprotinin is added to the serum.
7. Process for producing stabilized cytokeratin, wherein aprotinin is added to purified cytokeratin.
8. Use of aprotinin to stabilize cytokeratin.
9. Immunological method for determining cytokeratin in a sample, wherein the signal determined for the sample is compared with the signal that is obtained with the aid of the calibrator as claimed in claims 1 to 4, the calibrator being measured with the same method as for the sample.